Overlapping Signals for Protein Degradation and Nuclear Localization Define a Role for Intrinsic RAG-2 Nuclear Uptake in Dividing Cells

Identification of an NLS in the RAG-2 recombination-dispensable region. (A) Fluorescence confocal microscopy of NIH 3T3 fibroblasts transiently transfected with constructs expressing GFP alone (panels p to r) or GFP fusions to portions of RAG-2 (panels a to o). RAG-2 amino acid residues contained within each fusion protein are indicated at right. A20 mature B cells (B) and 63-12 pro-B cells (C) are shown. (D) NIH 3T3 cells transfected with GFP-LacZ (147 kDa) or GFP-LacZ fused to the 89 carboxy-terminal amino acids of the RAG-2 protein. In all cases DAPI is used as a counterstain for the nucleus, and representative cells are shown. Numbers indicate the percentage of cells demonstrating localization similar to those shown and correspond to Tables 1 to 3.

Identification of a RAG-2 nuclear localization sequence by clustered alanine scanning mutagenesis. Full-length GFP-RAG-2 fusion proteins bearing the indicated alanine scanning mutations were transfected into NIH 3T3 cells and localized by fluorescence microscopy. The wild-type (wt) RAG-2 amino acid sequence is given at top for residues 439 through 527; for each mutant protein, designated at left by an index letter (a through j), the positions of alanine substitutions are indicated (upper panel). The distributions of DAPI, GFP, or merged DAPI and GFP fluorescence (overlay) are shown in the lower panel. Representative cells are shown. Letters (a to j) correspond to individual mutant proteins as defined in the upper panel. Numbers in upper right corners of the overlay series indicate the percentages of cells with localization patterns similar to those shown.

RAG-1 and RAG-2 cooperate in localizing the V(D)J recombinase. NIH 3T3 cells were transfected with CFP-RAG-2 constructs (green pseudo-color) and YFP-RAG-1 constructs (red pseudo-color) and assayed by fluorescence microscopy. Representative cells are shown. (A) Wild-type YFP-RAG-1 was transfected either alone or in combination with CFP-fused RAG-2 proteins. DAPI, RAG-1 (YFP), or merged DAPI and YFP signals are displayed for transfection with RAG-1 alone (top panel). Coexpression patterns of YFP-RAG-1 and CFP fusions containing full-length RAG-2(1-527) or RAG-2(499/508A₁₀) are displayed in the lower panel. In each case, RAG-2, RAG-1, and merged signals are shown. Numbers indicate the percentages of cells positive for RAG-1 and RAG-2 and demonstrating RAG-2 localization patterns similar to those shown. The graph at right displays the localization pattern of RAG-2(499/508A₁₀) in the presence (solid bars) or absence (hatched bars) of wild-type RAG-1. (B) Transfection of YFP-RAG-1(BV) alone (top panel) or with CFP-RAG-2 (lower panel), displayed as in panel A. Numbers indicate the percentages of cells positive for RAG-1 and RAG-2 and demonstrating RAG-2 localization patterns similar to those shown. The graph at right displays localization pattern of RAG-2(499/508A₁₀) in the presence (solid bars) or absence (hatched bars) of RAG-1(BV). (C) Transfection of YFP-RAG-1(1/2BIV) either alone (top panel) or with CFP fused to wild-type RAG-2 (lower panel), displayed as in panel A. Numbers indicate the percentages of cells positive for RAG-1 and RAG-2 and demonstrating RAG-1 localization patterns similar to those shown. The graph at right displays localization pattern of RAG-1(1/2 BIV) in the presence (solid bars) or absence (hatched bars) of wild-type RAG-2.

Cell cycle-dependent impairment of V(D)J recombination by selective mutation of the RAG-2 nuclear localization sequence. Cells were transfected at either 20% confluency (A and C) or 90% confluency (B and D) with wild-type RAG-1, the indicated RAG-2 constructs, and the extrachromosomal substrate pJH200. At 36 h after transfection, cells were collected and assayed for rearrangement (A and B) or for DNA content by fluorescence-activated cell sorting (C and D). Complete data for recombination assays are listed at www.bs.jhmi.edu/mbg/RossEtAlAppendix.pdf . Representative fluorescence-activated cell sorter profiles are shown. Numbers indicate the percentages of cells with 2C DNA content.